Contaminating organisms in mass cultivation present one of the major challenges that must be overcome for successful commercialization of algal biofuels. The present study identified a range of contaminating organisms in Chlorella cultures cultivated in outdoor raceway ponds at the Arizona Center for Algae Technology and Innovation (Mesa, AZ). Nineteen organisms or Operational Taxonomic Units (OTU) in the Chlorella culture were identified by a combination of microscopic observation and 18S rRNA denaturing gradient gel electrophoresis (DGGE). More detailed analyses identified these contaminating organisms as 2 fungi, 7 flagellates, 3 amoebae, 4 ciliates, 1 rotifer, and 2 large insects. Among them Poterioochromonas sp., a small flagellate, appeared to be one of the most harmful causing culture collapse. In order to control Poterioochromonas sp., various operational parameters and application strategies of ultrasonic treatment were investigated. During sixteen-day consecutive Chlorella cultivation in batch mode, the ultrasonication conditions of 6 L min(-1) flow rate with the power of 495 W at 100% amplitude and a treatment frequency of once for 1 h every day, was proven to be the most effective in preventing Poterioochromonas outbreak in Chlorella culture with volume of 60 L. The above ultrasonication method was also effective at destroying an unknown fungus, an amoeba (Acanthocystis sp.), and ciliates (a member of the family Orchitophryidae). Our findings can serve as a technical foundation for the application of ultrasonication to control some of the contaminating microorganisms in mass cultivation of microalgae including Chlorella.